Biomarker for hepatolenticular degeneration diagnosis and liver-brain type differential diagnosis and test verification method of biomarker

Abstract

The invention provides a biomarker for hepatolenticular degeneration diagnosis and liver-brain type differential diagnosis. The biomarker comprises a serum neurofilament light chain protein NfL. The invention relates to a test verification method of a biomarker for hepatolenticular degeneration diagnosis and liver-brain type differential diagnosis, which comprises the following steps: test I, early diagnosis of Wilson disease by serum neurofilament light chain protein and superoxide dismutase, and liver-brain type differential diagnosis test: (1) collecting patient data, (2) detecting serum neurofilament light chain protein NfL, and (3) detecting serum superoxide dismutase SOD; (4) statistical analysis; and (5) test result obtaining. 5-1) general clinical data comparison. 5-2) expression difference of three groups of serum neurofilament light chain protein NfL levels; 5-3) expression difference of the three groups of serum superoxide dismutase (SOD) levels; 5-4) performing ROC curve analysis; and 5-5) analyzing the correlation between the serum NfL and SOD and the age, disease course time and UWDRS score. The serum NfL can be used as a biomarker for hepatolenticular degeneration diagnosis and liver-brain type differential diagnosis.

Description

Technical Field

[0001] The present invention relates to a biomarker for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type and a test verification method of the biomarker. Background Art

[0002] Hepatolenticular degeneration (HLD), also known as Wilson disease (WD), is an inherited, rare, progressively worsening copper (Cu) metabolism disorder. A retrospective study in 2020 showed that the prevalence of the disease is between 1:40,000 and 1:50,000. WD is caused by a genetic defect in the copper-transporting P-type ATPase (ATP 7B), which is mainly expressed in hepatocytes, but not limited to them. ATP 7B is mainly responsible for the excretion of copper in bile and the binding of copper to phospholipid proteins. ATP 7B inactivation leads to abnormal Cu deposition in the liver, causing hepatocyte damage and death, and releasing free, non-phospholipid-bound Cu into the blood circulation, which accumulates in different organs, leading to the complex clinical manifestations of WD. The most common clinical symptoms of WD mainly include liver disease, neurological deficits and mental disorders, among which liver diseases such as increased transaminase, liver failure, cirrhosis, etc. Precisely because it is a rare autosomal recessive genetic disease, the clinical manifestations are so hidden and diverse that diagnosis and treatment are often delayed. Studies have shown that with early diagnosis and appropriate treatment, nearly 85% of WD patients have good outcomes. The diagnosis of WD is mainly completed through serological data, physical examination results, clinical history, and molecular genetic testing. Early identification of WD remains challenging because the disease has a strong ability to mimic and requires a high degree of suspicion for correct and timely diagnosis. Current treatments include limiting dietary copper intake, reducing copper absorption with zinc, using chelation therapy to remove copper from tissues, and symptomatic treatment. Early detection of the disease indicates a better recovery prospect after treatment. Neurofilament protein (NF) is the main intermediate filament component of the neuronal cytoskeleton. It is organized into 10nm thick filaments in axons and dendrites. It is a large, heavily phosphorylated protein with multiple phosphate acceptor sites. It is mainly composed of four subunits including NfL, NfM, NfH and α-innexin or peripherin. Neurofilament protein is essential for the radial growth and structural stability of myelinated axons and for achieving the conduction velocity of axons. Compared with other subtypes, serum neurofilament light chain protein Neurofilament light chain protein, NfL has the characteristics of the lightest weight, the highest content, and the strongest solubility. Therefore, it is very important for the cell structure stability of neurons in the central nervous system CNS and the peripheral nervous system PNS. Due to the internal metabolism of the organism and exposure to toxic environmental substances and nitrogen species, it will be exposed to reactive oxidants, thereby generating reactive oxygen species, ROS, leading to oxidative stress.Oxidative stress induced by ROS is associated with various forms of disease, especially central nervous system diseases. Excessive ROS consumes endogenous antioxidants, which cannot offset all ROS, leading to cell damage. Superoxide dismutase (SOD) is one of the multiple defense mechanisms that prevents cell apoptosis caused by oxidative damage and plays a key role in alleviating oxidative stress. When the level of intracellular free Cu is too high, they promote the production of ROS through the Fenton reaction, increase the oxidative stress toxicity reaction, and lead to mitochondrial death. In traditional Chinese medicine, the pathogenesis of WD is mostly related to copper toxicity accumulation, phlegm and blood stasis, internal dampness and heat, and liver and kidney deficiency. Compared with Western medicine, traditional Chinese medicine plays its comprehensive treatment advantages of multi-target, multi-pathway, and less adverse reactions in the treatment of WD, but it is slow to take effect, has a long duration of action, and has a relatively weak copper removal effect. It is difficult for a single drug to exert a good therapeutic effect, so the combination of traditional Chinese and Western medicine has outstanding advantages in the treatment of WD. Serum NfL, as a highly specific axonal cytoskeleton protein, and SOD, as a key enzyme in the anti-oxidative stress response, are expected to be used in the diagnosis of WD patients, differential diagnosis of hepatocerebral type, and assessment of disease severity, which will help promote the research progress of WD. Serum NfL and SOD may be involved in the pathogenesis of WD, so it was previously speculated that they can be used for the diagnosis of WD patients, differential diagnosis of hepatocerebral type, and assessment of disease severity. However, at present, there is still controversy about whether serum NfL and SOD can be widely used in clinical practice. The standards for their peripheral blood and cerebrospinal fluid (CSF) levels have not yet been clarified, and it is still unknown whether the expression level has any significance for judging the progression between different types. Summary of the invention

[0003] Based on the above-mentioned defects in the prior art, the technical problem to be solved by the present invention is to provide a biomarker for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type, wherein the biomarker includes serum neurofilament light chain protein NfL.

[0004] As a further improvement of the present invention, the biomarker also includes superoxide dismutase SOD.

[0005] The present invention also provides a method for testing and verifying biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type, comprising:

[0006] Test 1: Serum neurofilament light chain protein and superoxide dismutase for early diagnosis of Wilson disease and differential diagnosis of hepatic and cerebrovascular type:

[0007] (1) Collect patient information

[0008] (2) Serum neurofilament light chain protein NfL detection

[0009] 2-1) Sample collection and preservation

[0010] All subjects were given 3-5 ml of fasting venous blood from patients and healthy subjects in the morning using serum tubes. The collected blood samples were placed at room temperature for 2 hours, then centrifuged at 1000×g for 20 minutes at 4°C, the upper suspended matter was removed, and the supernatant was placed at -80°C for testing. The serum samples were tested using the enzyme-linked immunosorbent assay (ELISA) method.

[0011] 2-2) Operation steps

[0012] The fasting venous blood of the patients and healthy subjects was centrifuged in the morning for testing. The specific testing steps are as follows:

[0013] ① Take out the required strips from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining strips in a ziplock bag and place them back at 4℃;

[0014] ② Sample addition: add 100 μL of samples or standards of different concentrations to the corresponding wells, add 100 μL of universal diluent to the blank wells, cover with sealing film and incubate at 37°C for 60 minutes;

[0015] ③ Add biotinylated antibody: Take out the enzyme label solution, discard the liquid, do not wash, directly add 100 μL of biotinylated antibody working solution to each well, cover with sealing film and incubate at 37°C for 60 minutes;

[0016] ④ Wash the plate: discard the liquid, add 300μL 1× washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 3 times;

[0017] ⑤ Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes;

[0018] ⑥ Wash the plate: discard the liquid and wash the plate 5 times according to the washing method in step ④;

[0019] ⑦ Add substrate: Add 90 μL of substrate TMB3,3',5,5'-tetramethylbenzidine to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes;

[0020] ⑧Add stop solution: Take out the ELISA solution or ELISA plate, add 50 μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm;

[0021] (3) Serum superoxide dismutase (SOD) detection;

[0022] 3-1) Sample collection and storage are the same as “2-1) Sample collection and storage”;

[0023] 3-4) Operation steps

[0024] The fasting venous blood of the patients and healthy subjects was centrifuged in the morning for testing. The specific testing steps are as follows:

[0025] ① Dilution and addition of standard: set 10 wells for standard, add 50μL of standard diluent, mix and dilute;

[0026] ② Sample addition: Set up blank wells and sample wells respectively. First add 40 μL of sample diluent to the sample wells on the enzyme-labeled plate, and then add 10 μL of the sample to be tested. The final dilution of the sample is 5 times;

[0027] ③ Add enzyme: add 50 μL of enzyme labeling reagent to each well, except for the blank well;

[0028] ④ Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes;

[0029] ⑤ Liquid preparation: dilute the 30 times concentrated washing solution with 30 times distilled water for later use;

[0030] ⑥ Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry;

[0031] ⑦ Color development: first add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color development at 37℃ in the dark for 10 minutes;

[0032] ⑧Stop: Add 50 μL of stop solution to each well to stop the reaction, and the blue color will turn yellow immediately;

[0033] ⑨ Use the blank well as zero setting, and measure the absorbance OD value of each well in sequence at a wavelength of 450nm;

[0034] (4) Statistical analysis

[0035] ① The experimental data were statistically analyzed using SPSS 26.0 statistical software: the independent sample t test was used to compare the two independent numerical variable data when they met the normal distribution; the chi-square test was used to analyze the correlation and difference of the completely random unordered categorical variable data. 2 test;

[0036] The two groups refer to the WD-brain type group and the WD-liver type group in the general data, which are two independent numerical variable data in the comparison of disease course; the WD-brain type group and the WD-liver type group in the general data are completely random and unordered categorical variable data in the comparison of gender;

[0037] ②When the three groups of samples meet normality, homogeneity of variance and independence at the same time, one-way analysis of variance is used;

[0038] If not satisfied, the Kruskal-Wallis H rank sum test was used;

[0039] ③ The receiver operating characteristic curve (ROC) analysis was used to evaluate the predictive efficacy of serum NfL and SOD expression levels in the diagnosis of WD patients and the differential diagnosis of hepatocerebral type, calculate the sensitivity and specificity, and evaluate the authenticity of the prediction;

[0040] (5) Test results

[0041] 5-1) Comparison of general clinical data

[0042] A total of 60 patients with Wilson's disease who met the inclusion criteria were selected, including 24 patients with hepatic type and 36 patients with brain type. During the process, there was no dropout in the hepatic type group, 2 patients in the brain type group, 1 patient withdrew from the experiment due to an accidental fall, and 1 patient refused to do a routine stool test; there were 30 healthy control groups with no dropouts; the actual enrolled patients were 24 patients in the WD-hepatic type group and 34 patients in the WD-brain type group; there were 16 males and 14 females in the healthy control group, with an average age of 27.43±5.66 years old; there were 19 males and 15 females in the WD-brain type group, with an average age of 28.72± 7.92 years old, with an average course of disease of 11.94±5.345 years; 13 males and 11 females in the WD-liver type group, with an average age of 27.67±8.53 years old and an average course of disease of 10.83±3.841 years; The chi-square test was used to analyze the gender of the three groups of patients and analyze their statistical significance. The age and course of disease were tested for normality and homogeneity of variance to determine whether the observation period met the normal distribution and homogeneity of variance. The age of the three groups was analyzed by one-way analysis of variance. The course of disease in the WD-liver type group and the WD-brain type group was tested by independent sample t test, and the results were listed and plotted at the same time;

[0043] 5-2) Differences in the expression of serum neurofilament light chain protein NfL among the three groups

[0044] The serum NfL levels among the three subgroups were statistically analyzed. First, a normality test was performed, and it was found that none of the three groups conformed to the normal distribution. Therefore, the Kruskal-Wallis H test was used to compare the three groups, including the comparison of relevant indicators among the three groups: WD-liver type, WD-brain type and healthy control group; then the Nemenyi test was used to perform post hoc comparisons between the two groups, and the tables and graphs were observed at the same time;

[0045] 5-3) Differences in the expression of serum superoxide dismutase (SOD) levels among the three groups

[0046] Similarly, the Kruskal-Wallis H test was used to detect the serum superoxide dismutase SOD levels of the three groups, and the results were tabulated and plotted;

[0047] 5-4) ROC curve analysis

[0048] In order to evaluate the value of the two indicators for the diagnosis of WD and the differential diagnosis of hepatocerebral type, ROC curve analysis was performed to observe the cutoff value, sensitivity, specificity, and area under the AUC curve of WD patients compared with the healthy control group, and the specific ROC curve of serum NfL level expression was plotted; including the cutoff value, sensitivity, specificity, and area under the AUC curve between WD hepatocerebral types, and the plot was also plotted;

[0049] If the statistical results have proved that there is no significant difference in serum SOD levels between WD brain-type patients and WD liver-type patients, the ROC curve diagram of serum SOD level expression can be drawn only for WD patients and healthy controls;

[0050] 5-5) Correlation analysis between serum NfL, SOD and age, disease duration and UWDRS score

[0051] If serum NfL does not follow a normal distribution, the Spearman correlation coefficient can be used to analyze the correlation between serum NfL and age, disease duration, and UWDRS score, and a table of correlations between serum NfL and age, disease duration, and UWDRS score can be plotted for analysis.

[0052] As a further improvement of the present invention: the (1) collecting patient information includes:

[0053] Name, gender, disease course, clinical symptoms, family history, laboratory test indicators, UWDRS scale test; observation indicators include: serum neurofilament light chain protein NfL, superoxide dismutase SOD.

[0054] As a further improvement of the present invention: 2-2), operation steps: the diluent is a universal diluent, 96-well configuration: 2×20mL; 48-well configuration: 1×20mL; storage conditions after opening: 2-8°C;

[0055] The concentrated biotinylated antibody 100× Biotin-antibody (100×) is 120 μL for 96-well configuration and 60 μL for 48-well configuration.

[0056] Preparation of the biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute, dilute the 100× concentrated biotinylated antibody to 1× working concentration with universal diluent, and prepare it for use immediately;

[0057] The concentrated enzyme binding working solution 100×Streptavidin-HRP (100×), 96-well configuration: 120 μL; 48-well configuration: 60 μL; storage conditions after opening: -20°C, away from light;

[0058] Preparation of the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute, dilute the 100× concentrated HRP enzyme conjugate to 1× working concentration with universal diluent, and prepare it for use immediately.

[0059] As a further improvement of the present invention, the universal diluent: 96-well configuration: 2×20 mL; 48-well configuration: 1×20 mL; storage conditions after opening: 2-8°C.

[0060] As a further improvement of the present invention: 3-4) in the operation step, the color developer solution is used as a substrate of horseradish peroxidase HRP in ELISA, and the color developer is TMB3,3',5,5'-tetramethylbenzidine.

[0061] The test method for biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type provided by the present invention also includes:

[0062] Experiment 2: Clinical observation on the effect of Bushen Huoxue Huazhuo prescription on the expression levels of serum NfL and SOD in patients with liver-kidney deficiency and phlegm-stasis syndrome

[0063] 1 Test subjects

[0064] 1.1 Case source

[0065] A total of 58 WD patients who met the inclusion criteria were selected and randomly divided into an observation group and a control group at a ratio of 1:1, with 29 cases in each group; the control group had 16 males and 11 females; the observation group had 14 males and 15 females;

[0066] 2 Test content

[0067] 2.1 Group treatment

[0068] Control group: 6 courses of conventional western medicine, 6 days per course, 2 days off: conventional copper excretion drugs: ① 5% glucose injection 250ml + dithiothiopropane sulfonate injection DMPS 0.02g / kg·d intravenous drip qd; ② 10% calcium gluconate injection 10ml + 5% glucose injection 50ml intravenous drip bid, used during the interval of DMPS withdrawal; ③ zinc gluconate tablets 280mg orally tid;

[0069] Observation group: conventional western medicine + Bushen Huoxue Huazhuo prescription, Bushen Huoxue Huazhuo prescription was used for 6 courses, each dose of crude drug weighed 200g, taken in the morning and evening: on the basis of the control group, Chinese medicine Bushen Huoxue Huazhuo prescription was added: Rehmannia glutinosa 20g, Morinda officinalis 15g, Schisandra chinensis 6g, Salvia miltiorrhiza 15g, Coptis chinensis 9g, Alisma orientalis 9g, Curcuma 9g, Rhubarb 6g, Lysimachia chinensis 15g, Curcuma longa 10g;

[0070] 2.2 Observation indicators

[0071] (1) Changes in clinical efficacy

[0072] The clinical efficacy of the two groups of patients after treatment was observed. According to the modified Goldstein clinical grading standard, the symptoms and signs were divided into markedly effective, improved, ineffective, and worsened.

[0073] (2) Changes in UWDRS scores

[0074] The Wilson disease rating scale was observed in both groups before and after treatment.

[0075] (3) Changes in TCM syndrome score

[0076] Observe the changes of TCM syndrome scores in the two groups before and after treatment;

[0077] (4) Laboratory examination

[0078] The patients' 24-hour urine copper, serum neurofilament light chain protein NfL, and superoxide dismutase SOD levels were tested before and after treatment. The specific operation methods for the determination of serum NfL and SOD levels are detailed in Experiment 1 (2) Serum neurofilament light chain protein NfL detection (3) Serum superoxide dismutase SOD detection;

[0079] (5) Safety indicators

[0080] Observe the patient's general examination items during the entire medication period, including basic vital signs, blood, urine, stool routine tests, and liver and kidney function, and record and report adverse events in a timely manner;

[0081] 2.3 Statistical analysis

[0082] SPSS 26.0 statistical software will be used for statistical analysis, and data will be expressed as x±s. When two groups of independent and continuous numerical variable data meet the normal distribution, the independent sample t test will be used. The statistical result P<0.05 indicates that the difference is statistically significant.

[0083] 3 Test results

[0084] 3.1 Comparison of general clinical data

[0085] A total of 58 patients with WD liver and kidney deficiency and phlegm and blood stasis were included, with 29 cases in the observation group and 29 cases in the control group. There were 16 males and 11 females in the control group, with an average age of 27.38±6.81 years; there were 14 males and 15 females in the observation group, with an average age of 28.24±8.20 years;

[0086] 3.2 Comparison of clinical efficacy between the two groups of patients after treatment

[0087] The overall effective rate of the observation group and the control group after the sixth course of treatment were compared; the P value of the two groups was compared by chi-square test;

[0088] 3.3 Comparison of UWDRS scores between the two groups

[0089] The UWDRS scores of the two groups of patients before and after treatment were compared;

[0090] 3.4 Comparison of TCM syndrome scores between the two groups

[0091] The TCM syndrome score values ​​of the two groups of patients were observed before the experiment and after treatment. The TCM syndrome score values ​​of the two groups were compared after treatment, and the TCM syndrome score values ​​of the Bushen Huoxue Huazhuo prescription combined with western medicine group were observed.

[0092] 3.5 Comparison of 24-hour urinary copper between the two groups

[0093] Compare the urinary copper in the control group and the Bushen Huoxue Huazhuo prescription combined with western medicine group before treatment, and the difference of urinary copper between the two groups after treatment;

[0094] 3.6 Comparison of serum neurofilament light chain protein NfL between the two groups of patients

[0095] Comparison of serum NfL values ​​between the two groups before treatment; Comparison of serum NfL values ​​between the observation group and the control group after treatment; Comparison of serum NfL values ​​between the two groups after treatment, between the Bushen Huoxue Huazhuo prescription combined with western medicine group and the control group;

[0096] 3.7 Comparison of serum superoxide dismutase (SOD) between the two groups

[0097] The difference in serum SOD between the two groups of patients before treatment was observed after statistical processing; the serum SOD level of the observation group after treatment, the serum superoxide dismutase SOD level of the control group after treatment; the two groups were compared after treatment, and the difference between the Bushen Huoxue Huazhuo prescription combined with western medicine group and the control group was compared.

[0098] As a further improvement of the present invention: when testing the serum NfL level, the whole blood sample collected in the serum separation tube is placed at room temperature for 2 hours or at 2-8°C overnight, and then centrifuged at 1000×g for 20 minutes, and the supernatant is taken, or the supernatant is stored at -20°C or -80°C, but repeated freezing and thawing should be avoided;

[0099] Preparation work before testing serum NfL levels: add 1mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved, then gently mix it, the concentration is 500pg / mL, and finally determine the following concentrations: 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.2pg / mL, 15.6pg / mL, 7.8pg / mL, 0pg / mL for dilution, take 7 EP centrifuge tubes, add 500μL of universal diluent to each tube, draw 500μL from the 500pg / mL standard working solution into the first EP tube and mix it to make a 250pg / mL standard working solution, and follow this step to draw and mix in sequence; the last tube is directly used as a blank well, and there is no need to draw liquid from the second to last tube;

[0100] During the sample addition process for serum NfL level detection, samples or standards of different concentrations were added to the corresponding wells at 100 μl per well, and 100 μl of universal diluent was added to the blank wells.

[0101] Compared with the prior art, the present invention has the following technical effects:

[0102] The value of serum neurofilament light chain protein and superoxide dismutase in the early diagnosis of Wilson disease and differential diagnosis of hepatocerebral type: ① Comparison of general clinical data: There were no significant differences in age, gender, and disease course of patients with WD hepatocerebral type among the three groups (P>0.05); ② Differences in serum NfL levels among the three groups: The serum NfL level of patients with WD cerebral type [44.44 (38.99-48.71) pg / mL] was significantly higher than that of patients with WD hepatic type [29.20 (17.54-42.48) pg / mL; P<0.01] and healthy control group [12.82 (4.825-17.58) pg / mL, P<0.01], and the serum NfL expression level of patients with WD hepatic type was significantly higher than that of the control group (P<0.01); ③ Differences in serum SOD levels among the three groups: WD cerebral type [152.48 (146.09-1 The serum SOD expression levels in patients with WD brain type [62.22) U / mL] and WD liver type [148.49 (137.57-159.24) U / mL] were significantly lower than those in the healthy control group [194.08 (173.86-215.17) U / mL, P < 0.01], while there was no significant difference in the serum SOD expression levels between patients with WD brain type and WD liver type (P > 0.05); ④ ROC curve analysis: In terms of serum NfL expression levels, compared with the healthy control group, the cutoff value of WD patients was 23.925 pg / mL, the sensitivity was 87.9%, the specificity was 90%, and the area under the AUC curve was 0.9121 (P < 0.05); between WD liver and brain type, the cutoff value was 33.355 pg / mL, the sensitivity was 94.1%, the specificity was 75%, and the area under the AUC curve was 0.773 (P < 0.05). In terms of serum SOD expression level, compared with the healthy control group, the cutoff value of WD patients was 169.025 U / mL, the sensitivity was 87.9%, the specificity was 80%, and the area under the AUC curve was 0.8839 (P < 0.05); ⑤ Correlation between serum NfL, SOD and age, disease course and UWDRS score: There was no significant correlation between serum NfL and SOD and age or disease course (P > 0.05), the serum NfL expression level was significantly positively correlated with the UWDRS score, and the serum SOD expression level was significantly negatively correlated with the UWDRS score.

[0103] Clinical efficacy observation of the Bushen Huoxue Huazhuo prescription on the expression levels of serum NfL and SOD in patients with liver-kidney deficiency and phlegm-stasis syndrome WD: ① Comparison of the effective rates between the two groups: After the sixth course of treatment, the overall effective rate of the observation group was 79.31%, and the overall effective rate of the control group was 51.32%. Both treatment methods were effective for patients with liver-kidney deficiency and phlegm-stasis syndrome WD, but the overall efficacy of the Bushen Huoxue Huazhuo prescription combined with western medicine group was better than that of the control group (P < 0.05); ② Comparison of TCM syndrome scores between the two groups: After treatment, the TCM syndrome scores of the two groups of patients decreased (P < 0.05), and the TCM syndrome scores of the Bushen Huoxue Huazhuo prescription combined with western medicine group decreased more significantly (P = 0.0036 < 0.01); ③ Comparison of UWDRS scores between the two groups: After treatment, the UWDRS scores of the control group and the Bushen Huoxue Huazhuo prescription combined with western medicine group were significantly reduced (P < 0.01) ④Comparison of 24-hour urinary copper between the two groups: After treatment, the 24-hour urinary copper excretion of both groups increased (P<0.05), and the BUSHENHUOXUEHUAZHUO prescription combined with western medicine group was more effective in promoting urinary copper excretion than the simple western medicine group (P<0.05); ⑤Comparison of serum NfL levels between the two groups: The serum NfL levels of the control group and the BUSHENHUOXUEHUAZHUO prescription combined with western medicine group were significantly decreased compared with those before treatment (P<0.01); After treatment, the serum NfL values ​​of the BUSHENHUOXUEHUAZHUO prescription combined with western medicine group decreased more significantly than those of the control group (P<0.01); ⑥Comparison of serum SOD levels between the two groups: The serum SOD expression levels of both groups increased after treatment compared with those before treatment (P<0.05), and the increase was more significant in the BUSHENHUOXUEHUAZHUO prescription combined with western medicine group (P<0.01). BRIEF DESCRIPTION OF THE DRAWINGS

[0104] Figure 1 Yes: Comparative chart of clinical and general data in trial one.

[0105] Figure 2 It is: The difference in expression of serum NfL levels among the three groups in Experiment 1.

[0106] Figure 3 It is: The difference diagram of serum SOD level expression among three groups in experiment one.

[0107] Figure 4 Yes: ROC curve of serum NfL level expression in experiment one.

[0108] Figure 5 Yes: ROC curve diagram of serum SOD level expression in experiment one.

[0109] Figure 6 Yes: Correlation graph between serum NfL and age, disease duration, and UWDRS score in Experiment 1.

[0110] Figure 7It is: the correlation diagram between serum SOD and age, disease duration and UWDRS score in experiment 1.

[0111] Figure 8 Yes: Comparison chart of general clinical data in Experiment 2.

[0112] Fig. 9 Yes: Comparison of the overall effective rates between the observation group and the control group in Experiment 2.

[0113] Fig.10 Yes: Comparison of UWDRS scores of the two groups of patients in Experiment 2 before and after treatment.

[0114] Fig.11 Yes: Comparison of TCM syndrome integral values ​​of the two groups of patients in Experiment 2 before and after treatment.

[0115] Fig.12 Yes: Comparison of 24h urinary copper (μg) in the two groups of patients in Experiment 2 before and after treatment.

[0116] Fig.13 Yes: Comparison of serum NfL (pg / mL) levels in the two groups of patients in Trial 2 before and after treatment.

[0117] Fig.14 It is: Comparison of serum SOD (U / mL) levels of the two groups of patients in Experiment 2 before and after treatment.

[0118] Fig.15 Yes: According to the operation steps in 2.2.4, the fasting venous blood of the patients and healthy subjects was centrifuged in the morning for examination. The specific test steps are shown in the figure.

[0119] Fig.16 Yes: According to the operation steps in 2.3.4, the fasting venous blood of patients and healthy subjects was centrifuged in the morning for examination. The specific test steps are shown in the figure.

[0120] in, Fig. 9 In the middle, the markedly effective, improved, ineffective and worsening groups correspond to the indicated positions, the left is the control group, and the right is the observation group; Figure 10-14 Middle: The corresponding position before treatment (left) is the control group, and the corresponding position before treatment (right) is the observation group; the corresponding position after treatment (left) is the control group, and the corresponding position after treatment (right) is the observation group; in addition, in the upper right position of each figure, the control group is shown in green, and the observation group is shown in red. DETAILED DESCRIPTION

[0121] The following is a further and more detailed description of a biomarker for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type provided by the present invention and a test verification method of the biomarker through specific examples:

[0122] The present invention intends to evaluate the potential of serum NfL and SOD as biomarkers of WD disease by analyzing the expression levels of serum NfL and SOD in WD patients. The breakthrough of serum NfL as a highly specific axonal cytoskeletal protein and SOD as a key enzyme of anti-oxidative stress response is used to verify the above two indicators as biomarkers for the diagnosis of hepatolenticular degeneration, differential diagnosis of hepatocerebral type and its risk assessment value. The clinical efficacy of the Bushen Huoxue Huazhuo formula on hepatolenticular degeneration of liver and kidney deficiency and phlegm and blood stasis type is further explored based on the patient's serum NfL, SOD levels and 24h urine copper changes, in order to further explore the new small molecule mechanism of the Bushen Huoxue Huazhuo formula on nerve and liver protection, which has distinct characteristics of traditional Chinese medicine.

[0123] When testing serum NfL levels, due to different sample processing and requirements, it is necessary to perform multiple tests according to the test requirements and then collect accurately. Finally, a more accurate collection idea was obtained. The whole blood sample collected in the serum separation tube was placed at room temperature for 2 hours or 2-8°C overnight, then centrifuged at 1000×g for 20 minutes, and the supernatant was taken, or the supernatant was stored at -20°C or -80°C, but repeated freezing and thawing should be avoided.

[0124] In the preparation work before the serum NfL level test, the standard gradient working solution was not successfully prepared several times. After discussion, it was found that the concentration dilution gradient was inaccurate. Add 1mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved and gently mix (concentration is 500pg / mL), and finally determine the following concentrations: 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.2pg / mL, 15.6pg / mL, 7.8pg / mL, 0pg / mL for dilution. Take 7 EP tubes, add 500μL of universal diluent to each tube, draw 500μL from the 500pg / mL standard working solution into the first EP tube and mix to make a 250pg / mL standard working solution, and follow this step to draw and mix in sequence. The last tube is directly used as a blank well, and there is no need to draw liquid from the second to last tube.

[0125] During the serum NfL level test, samples or standards of different concentrations were added to the corresponding wells at 100 μl per well, and 100 μL of universal diluent was added to the blank wells. After covering the plate with the sealing film and incubating at 37°C for 60 minutes, it was found that the matrix effect affected the test results, so the samples to be tested were diluted again with universal diluent at least 1 times before adding them to the ELISA plate for testing. In order to reduce the impact of the matrix effect on the test results, the sample concentration should be multiplied by the corresponding dilution factor when calculating the sample concentration. It is recommended to set up duplicate wells for all samples and standards to be tested.

[0126] During the test, there were problems of weak or colorless color. After discussion, the following possible problems were summarized: insufficient incubation time, incorrect incubation temperature, insufficient reagent volume, incorrect dilution, and inactivation of enzyme conjugates. Then we ensured the incubation time, incubated at the recommended temperature, checked the pipette and strictly followed the operating procedures, checked the reagent dilution steps and mixed enzyme conjugates and substrates, and finally obtained more accurate results through color reaction inspection. During the serum SOD level detection and determination, several cases of low OD values ​​occurred. After discussion, the following possible problems were summarized: incorrect microplate reader settings, no stop solution, too long waiting time when reading the plate, and too high or too low sample content. Then in our experiment, we repeatedly checked the instrument wavelength, added an appropriate amount of stop solution, read the plate in time, and determined the appropriate dilution multiple through preliminary experiments, and finally obtained accurate OD values. In addition, after covering the plate with a sealing film and incubating at 37°C for 60 minutes, it was found that the matrix effect affected the test results, so the sample to be tested was diluted to a minimum of 1 times with a universal diluent before adding it to the microplate for testing.

[0127] Experiment 1: Study on the value of serum neurofilament light chain protein and superoxide dismutase in the early diagnosis and differential diagnosis of Wilson disease

[0128] 1 Test method

[0129] 1.1 Case source

[0130] A total of 60 WD patients who met the inclusion criteria were selected, including 36 cases in the brain type group and 24 cases in the liver type group. All cases were patients hospitalized in the Brain Disease Center of Anhui Provincial Hospital of Traditional Chinese Medicine from January to December 2023; in addition, 30 healthy volunteers were included in accordance with the principle of voluntariness and after examination of serum ceruloplasmin, copper oxidase, etc., Wilson's disease and other liver diseases, nervous system diseases, etc. were excluded.

[0131] 1.2 Diagnostic criteria

[0132] 1.2.1 Diagnostic criteria for WD

[0133] According to the "Guidelines for the Diagnosis and Treatment of Hepatolenticular Degeneration in China 2021", the diagnostic criteria for WD are as follows: ① Extrapyramidal symptoms and (or) psychiatric symptoms with limb tremor, dystonia, bradykinesia, etc. as the main manifestations, as well as liver damage manifestations such as unexplained elevated transaminases and liver pain; ② Decreased serum ceruloplasmin and (or) elevated 24-hour urine copper; ③ Positive corneal KF ring (Kayser-Fleischer Ring); ④ Family history. Wilson disease can be diagnosed when ①+②+③ or ①+②+④ are met, and ATP7B gene testing can be performed if necessary.

[0134] 1.2.2 WD clinical classification criteria

[0135] According to the "Guidelines for the Diagnosis and Treatment of Hepatolenticular Degeneration in China 2021", the specific classifications are as follows: ① Liver type: symptoms of liver damage such as elevated transaminases, hepatitis, and cirrhosis. ② Brain type: extrapyramidal manifestations such as tremor, bradykinesia, ataxia, and dysarthria. ③ Other types: mainly manifested by kidney damage, bone and joint muscle damage, hemolytic anemia, etc. ④ Mixed type: a combination of the above types.

[0136] 1.3 Case inclusion criteria

[0137] ① Must comply with the clinical diagnostic criteria for WD in the 2021 Chinese Guidelines for the Diagnosis and Treatment of Wilson's Disease;

[0138] ② The patients must meet the liver type and brain type classification in the "2021 Chinese Guidelines for the Diagnosis and Treatment of Hepatolenticular Degeneration";

[0139] ③Age 10-49 years old (inclusive);

[0140] ④ The patient's condition is relatively stable, he has a certain level of education, and he can cooperate with examinations throughout the treatment process, including serological tests and UWDRS scale score measurements.

[0141] 1.4 Case Exclusion Criteria

[0142] ① Patients who do not meet the Western medical diagnostic criteria for WD, as well as the liver type and brain type in the clinical classification criteria;

[0143] ②Exclude extrapyramidal symptoms caused by cerebrovascular disease, drugs or other reasons;

[0144] ③ Exclude liver damage caused by other reasons such as alcoholic liver disease, autoimmune liver disease, viral hepatitis, etc.;

[0145] ④Other diseases related to serum NfL and SOD, such as Parkinson's disease, amyotrophic lateral sclerosis, cerebral hemorrhage, etc.;

[0146] ⑤WD is combined with severe mental symptoms;

[0147] ⑥ Suffering from other major life-threatening diseases, such as hepatic encephalopathy, upper gastrointestinal bleeding, brain tumor, etc.;

[0148] ⑦ Pregnant or lactating women;

[0149] ⑧ Those who cannot cooperate with the inspection for a long time.

[0150] 1.5 Inclusion criteria for the healthy group

[0151] ①Age: 18-50 years old;

[0152] ②No functional or organic diseases;

[0153] ③ Never used antibiotics, liver protection, nerve nutrition and other drugs in the past six months.

[0154] 1.6 Ethics

[0155] This study has been reviewed and approved by the Ethics Committee of the First Affiliated Hospital of Anhui University of Chinese Medicine (approval number: 2022MCZQ18).

[0156] 2 Test content

[0157] 2.1 Collection of general and clinical data

[0158] The collected patient data mainly include: name, gender, course of disease, clinical symptoms, family history, laboratory test indicators, UWDRS scale test, etc. The observation indicators mainly include: serum NfL, SOD, etc.

[0159] 2.2 Serum NfL detection

[0160] 2.2.1 Sample collection and storage

[0161] On the second day after admission, all subjects used ordinary serum tubes (red-headed tubes) to collect 3-5 ml of fasting venous blood from patients and healthy subjects in the morning. The collected blood samples were placed at room temperature for 2 hours and then centrifuged at 1000×g for 20 minutes at 4°C. The upper suspended matter was removed and the supernatant was placed at -80°C for testing. The serum specimens were tested by enzyme linked immunosorbent assay (ELISA) on the Redu automatic enzyme immunoassay analyzer according to the instructions of the kit and the concentration was calculated.

[0162] 2.2.2 Main reagents

[0163] This experiment uses the human serum neurofilament light chain protein (NfL) ELISA kit (Cat. No.: JL47298, Batch No.: 20231216, Manufacturer: Jianglai Biology) to detect serum NfL. The kit mainly includes the following items:

[0164]

[0165] 2.2.3 Instruments and equipment

[0166]

[0167]

[0168] 2.2.4 Operation steps

[0169] The fasting venous blood of the patients and healthy subjects was centrifuged in the morning for testing. The specific testing steps are as follows. Fig.15As shown. The standard substances used are composed of 2 for 96-well configuration and 1 for 48-well configuration. Standard substances are a series of antibody standard solutions with known concentrations, which are used to establish a standard curve to help determine the antibody concentration of the sample. The concentration of the standard substances is usually accurately measured to ensure the accuracy and comparability of the experimental results. This method is mainly used to detect antigens. Its basic principle is to connect specific antibodies to solid phase carriers to form solid phase antibodies. Subsequently, the standard substance of the sample to be tested is added to make it contact with the solid phase antibody for a period of time, so that the antigen in the sample binds to the antibody on the solid phase carrier to form a solid phase antigen complex. The purpose of this step is to allow the antigen to specifically bind to the antibody on the solid phase carrier to form a visible immune complex, which is convenient for subsequent detection and analysis. The enzyme label solution specifically refers to the enzyme label plate, and each well refers to the above sample. 300μL 1× washing solution is the standard ratio of 300μL 1× washing solution. Preparation of 1× washing solution: Take 10ml of 20× washing solution into 190ml of distilled water. The concentrated washing solution taken out from the refrigerator may have crystals, which is a normal phenomenon. It can be placed at room temperature and prepared after the crystals are completely dissolved. The stop solution is generally made of strong acid or strong base as the main formula. The above stop solution is 2m HCl and 2m H2SO4. The stop solution is a safe, ready-to-use acidic solution that does not contain sulfuric acid and is used to terminate the TMB color development reaction. The Chinese term for 100×Streptavidin-HRP is horseradish peroxidase labeled streptavidin.

[0170] 2.3 Serum SOD detection

[0171] 2.3.1 Sample collection and storage

[0172] Same as “2.2.1 Sample collection and preservation”.

[0173] 2.3.2 Main reagents

[0174] This experiment uses human superoxide dismutase (SOD) ELISA assay kit (Cat. No.: JYM2065Hu, Batch No.: 20240110, Manufacturer: Wuhan Gene Beauty Technology Co., Ltd.) to detect serum SOD levels. The kit mainly includes:

[0175]

[0176]

[0177] 2.3.3 Instruments and equipment Same as “2.2.3 Instruments and equipment”

[0178] 2.3.4 Operation steps

[0179] The fasting venous blood of the patients and healthy subjects was centrifuged in the morning for testing. Fig.16 shown.

[0180] 2.4 Statistical analysis

[0181] ① The data of this experiment were statistically analyzed using SPSS26.0 statistical software. The independent sample t test was used to compare the data of two independent numerical variables if they met the normal distribution. The chi-square test was used to analyze the correlation and difference of completely random unordered categorical variable data. 2 test.

[0182] ② When the three groups of samples simultaneously meet the normality, homogeneity of variance, and independence, one-way analysis of variance (ANOVA test) is used; if not, the Kruskal-Wallis H rank sum test is used.

[0183] ③The receiver operating character curve (ROC) analysis was used to evaluate the predictive efficacy of serum NfL and SOD expression levels in the diagnosis of WD patients and the differential diagnosis of hepatocerebral type, and the sensitivity and specificity were calculated to evaluate the authenticity of the prediction.

[0184] 3 Test results

[0185] 3.1 Comparison of general clinical data of research subjects

[0186] A total of 60 WD patients who met the inclusion criteria were selected in this study, including 24 liver type and 36 brain type. During the process, there was no dropout in the liver type group, 2 patients dropped out of the brain type group, 1 patient accidentally fell and withdrew from the experiment, and 1 patient refused to do a routine stool test. There were 30 healthy control groups, and no dropout. The actual number of patients enrolled was 24 in the WD-liver type group and 34 in the WD-brain type group. There were 16 males and 14 females in the healthy control group, with an average age of 27.43±5.66 years. There were 19 males and 15 females in the WD-brain type group, with an average age of 28.72±7.92 years and an average course of disease of 11.94±5.345 years. There were 13 males and 11 females in the WD-liver type group, with an average age of 27.67±8.53 years and an average course of disease of 10.83±3.841 years. The chi-square test was used to analyze the gender of the three groups of patients, and there was no statistically significant difference (P=0.978>0.05). The age and course of disease were tested for normality and variance homogeneity, and they all met the normal distribution and variance homogeneity. The age of the three groups was analyzed by one-way analysis of variance, and the course of disease of the WD-liver type group and the WD-brain type group was tested by independent sample t test, and the results showed no significant difference (P>0.05). See Table 1 for details. Figure 1 Note: ns means no statistical difference.

[0187] Table 1 Comparison of clinical and general data

[0188]

[0189] 3.2 Differences in serum NfL levels among the three groups

[0190] Statistical analysis was performed on the serum NfL levels among the three subgroups. First, a normality test was performed, and it was found that none of the three groups conformed to the normal distribution. Therefore, we used the Kruskal-Wallis H test to compare the three groups, and then used Nemenyi to perform post hoc comparisons between the two groups. The results showed that the serum NfL levels of patients with WD brain type [44.44 (38.99-48.71) pg / mL] were significantly higher than those of patients with WD liver type [29.20 (17.54-42.48) pg / mL; P < 0.01] and the healthy control group [12.82 (4.825-17.58) pg / mL, P < 0.01], and the serum NfL levels of patients with WD liver type were significantly higher than those of the control group (P < 0.01). See Table 2 for details. Figure 2 .

[0191] Table 2 Differences in serum NfL levels among three groups

[0192]

[0193] Note: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0194] 3.3 Differences in serum SOD levels among the three groups

[0195] Similarly, the Kruskal-Wallis H test was used to detect the serum SOD levels of the three groups. The results showed that the serum SOD levels of WD brain type [152.48 (146.09-162.22) U / mL] and liver type patients [148.49 (137.57-159.24) U / mL] were significantly lower than those of the healthy control group [194.08 (173.86-215.17) U / mL, P < 0.01], while there was no significant difference in serum SOD levels between WD brain type patients and WD liver type patients (P > 0.05). See Table 3 for details. Figure 3 .

[0196] Table 3 Differences in serum SOD levels among three groups

[0197]

[0198] Note: * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001.

[0199] 3.4 ROC curve analysis

[0200] In order to evaluate the value of the two indicators for the diagnosis of WD and differential diagnosis of hepatocerebral type, ROC curve analysis was performed. Compared with the healthy control group, the cutoff value of WD patients was 23.925 pg / mL, the sensitivity was 87.9%, the specificity was 90%, and the area under the AUC curve was 0.9121 (P < 0.05). Figure 4 a; Between WD hepatocerebral type, the cutoff value was 33.355 pg / mL, the sensitivity was 94.1%, the specificity was 75%, and the area under the AUC curve was 0.773 (P < 0.05). Figure 4 b.

[0201] Since the above statistical results have proved that there is no significant difference in serum SOD levels between WD brain type patients and WD liver type patients, only the ROC curve of WD patients and healthy controls was drawn. Compared with the healthy control group, the cutoff value of WD patients was 169.025 U / mL, the sensitivity was 87.9%, the specificity was 80%, and the area under the AUC curve was 0.8839 (P < 0.05). Figure 5 Note: a: healthy control group vs WD group; b: WD-brain type group vs WD-liver type group. 3.5 Correlation analysis between serum NfL, SOD and age, disease duration and UWDRS score

[0202] Since serum NfL does not follow a normal distribution, the Spearman correlation coefficient was used to analyze the correlation between serum NfL and age, disease duration, and UWDRS score. The results are as follows: NfL expression has no correlation with age (P = 0.56 > 0.05), no correlation with disease duration (P = 0.420 > 0.05), and is significantly positively correlated with UWDRS score (P < 0.01). See Table 4 for details. Figure 6 .

[0203] Table 4 Correlation between serum NfL and age, disease duration, and UWDRS score

[0204]

[0205]

[0206] Note: * indicates P < 0.05, the difference is statistically significant.

[0207] At the same time, the correlation between serum SOD and age, disease duration and UWDRS score was analyzed. The results were as follows: SOD expression was not correlated with age (P = 0.892 > 0.05), was not correlated with disease duration (P = 0.450 > 0.05), and was significantly negatively correlated with UWDRS score (P < 0.05). See Table 5 for details. Figure 7 .

[0208] Table 5 Correlation between serum SOD and age, disease duration, and UWDRS score

[0209]

[0210] Note: * indicates P < 0.05, the difference is statistically significant.

[0211] 4 Discussion

[0212] 4.1 Completion of the test

[0213] A total of 60 WD patients who met the inclusion criteria were selected in this study, including 24 liver-type patients and 36 brain-type patients. During the process, there was no dropout in the liver-type group, and 2 patients dropped out in the brain-type group. The actual number of patients enrolled was 24 liver-type patients and 34 brain-type patients. At the same time, 30 healthy controls were recruited, and no dropouts were found. The completion rate of the liver-type group and the healthy control group was 100%, and the completion rate of the brain-type group was 94.12%.

[0214] 4.2 Correlation analysis between serum NfL level and diagnosis and differential diagnosis of WD

[0215] Neurofilament is a cytoskeletal protein specifically expressed on neurons. Its abnormal increase can be caused in various pathological processes such as neurodegeneration, inflammatory response, and vascular disease. NfL is the lightest and most soluble protein among neurofilament proteins. It is released into the extracellular space after axonal damage, resulting in decreased protein expression and smaller axon diameter. It is the most widely studied neurofilament protein.

[0216] The experiment found that the serum NfL level of WD brain type patients with neurological manifestations [44.44 (38.99-48.71) pg / mL] was significantly higher than that of WD liver type patients [29.20 (17.54-42.48) pg / mL; P < 0.01] and healthy controls [12.82 (4.825-17.58) pg / mL, P < 0.01]. The serum NfL level of WD liver type patients was also significantly higher than that of the control group (P < 0.01). The ROC curve showed that compared with the healthy control group, the cutoff value of WD patients was 23.925 pg / mL, the sensitivity was 87.9%, the specificity was 90%, and the area under the AUC curve was 0.9121; the cutoff value of WD hepatocerebral type was 33.355 pg / mL, the sensitivity was 94.1%, the specificity was 75%, and the area under the AUC curve was 0.773 (P < 0.05), which showed that serum NfL can not only be used as a diagnostic marker for WD disease, but also as one of the effective indicators for differential diagnosis of hepatocerebral type. In addition, this study also compared the correlation between serum NfL and age, disease course and UWDRS score, and found that the expression of serum NfL was not correlated with age (P = 0.056), and was not correlated with the course of disease (P = 0.420), but was significantly positively correlated with the UWDRS score, indicating that serum NfL is related to the severity of the disease in WD patients.

[0217] 4.3 Correlation analysis between SOD level and WD diagnosis and differential diagnosis

[0218] Copper (Cu) homeostasis is essential for cell growth, differentiation, and tissue repair. When intracellular free Cu levels are excessive, they promote the generation of reactive oxygen species (ROS) via the Fenton reaction, which can lead to disease onset and progression or cell death. Oxidative stress triggered by ROS can cause damage to lipids, proteins, and DNA, and is therefore associated with countless pathologies. To limit ROS toxicity, cells use Cu-dependent chaperones, Cu-binding ceruloplasmin, and Cu-regulated enzymes such as SODs (mainly SOD1 and SOD3) to inhibit the production of Cu-favoring oxidative stress. + The major role of SOD in all aerobic organisms is to neutralize superoxide anion free radicals, which are generated in the cytosol, mitochondria, and endoplasmic reticulum of cells by dismutation into hydrogen peroxide and oxygen.

[0219] This study found that the serum SOD levels of WD brain type [152.48 (146.09-162.22) U / mL] and liver type patients [148.49 (137.57-159.24) U / mL] were significantly lower than those of the healthy control group [194.08 (173.86-215.17) U / mL, P < 0.01], while there was no significant difference in serum SOD levels between WD brain type patients and WD liver type patients (P > 0.05). The ROC curve results showed that compared with the healthy control group, the cutoff value of WD patients was 169.025 U / mL, the sensitivity was 87.9%, the specificity was 80%, and the area under the AUC curve was 0.8839 (P > 0.05). The SOD expression level was not correlated with age and disease course, but was significantly negatively correlated with the UWDRS score (P < 0.05). This indicates that serum SOD can only be used as a diagnostic marker for WD disease, and as the disease worsens, its expression level decreases, and it cannot be used as an effective biomarker for differential diagnosis of hepatocerebral type.

[0220] Experiment 2: Clinical observation on the effect of Bushen Huoxue Huazhuo prescription on the expression levels of serum NfL and SOD in patients with liver-kidney deficiency and phlegm-stasis syndrome

[0221] 1 Test method

[0222] 1.1 Case source

[0223] A total of 58 WD patients who met the inclusion criteria from the Brain Disease Center of Anhui Provincial Hospital of Traditional Chinese Medicine from January 2023 to December 2023 were selected and randomly divided into an observation group and a control group in a 1:1 ratio of 29 cases each. There were 16 males and 11 females in the control group; there were 14 males and 15 females in the observation group.

[0224] 1.2 Diagnostic criteria

[0225] 1.2.1 Diagnostic criteria for WD

[0226] Same as Part 1 “1.2.1 WD diagnostic criteria”.

[0227] 1.2.2WD TCM Syndrome Diagnostic Criteria

[0228] The TCM syndrome differentiation of the patient was determined by referring to the Diagnosis and Treatment Program of Hepatolenticular Degeneration edited by Yang Wenming et al. TCM syndrome differentiation was performed by two (deputy) chief TCM physicians and their opinions were consistent.

[0229] ① Phlegm and blood stasis syndrome: dull expression, decreased reaction, slurred speech, involuntary tremor of limbs, slow body movement, heartburn and nausea, drooling, chest and rib fullness, lumps under the ribs, rough skin or ecchymosis, pain when touched, dark tongue with ecchymosis, thin and greasy fur, stringy and slippery pulse. ② Liver and kidney deficiency syndrome: tremor of limbs, slow flexion and extension, knee contracture and toes retraction, clumsy walking, dull expression, insomnia and dreaminess, sore waist and legs, flank pain, fever in the five hearts, irritability, dry and red tongue with little fur, stringy and fine pulse.

[0230] 1.3 Case inclusion criteria

[0231] ① Must comply with the clinical diagnostic criteria for WD in the 2021 Chinese Guidelines for the Diagnosis and Treatment of Wilson's Disease;

[0232] ② The patients must meet the TCM disease diagnosis criteria of the Diagnosis and Treatment Program for Wilson's Disease and meet the diagnostic criteria for liver-kidney deficiency and phlegm-stasis syndrome; ③ The patients must be aged 10-49 years (inclusive); ④ The patients' condition is relatively stable, they have a certain level of education, and they can cooperate with examinations throughout the treatment process, including serological tests and UWDRS scale scores.

[0233] 1.4 Case Exclusion Criteria

[0234] ① Patients who do not meet the Western medical diagnostic criteria for WD, and whose TCM syndromes are not liver-kidney deficiency or phlegm-stasis syndrome;

[0235] ② Patients taking other Chinese medicine preparations or Chinese patent medicines; ③ Patients who fail to take medicine regularly. The rest is the same as "Part I 1.4 Case Exclusion Criteria ④, ⑤, ⑥, ⑦, ⑧".

[0236] 1.5 Case dropout and exclusion criteria

[0237] ① Patients who experience severe adverse reactions due to the drugs used during the study;

[0238] ② Those who fail to take Chinese medicine compound treatment according to the protocol, which affects the efficacy or safety assessment;

[0239] ③ Patients who exit the trial before completing the treatment due to various other reasons.

[0240] 2 Test content

[0241] 2.1 Group treatment

[0242] Control group: conventional western medicine (6 courses of treatment): conventional copper-excreting drugs were used: ① 5% glucose injection 250ml + sodium dithiosulfate injection (DMPS) 0.02g / (kg·d) intravenous drip qd, each course of treatment is 6 days, and the drug is stopped for 2 days; ② 10% calcium gluconate injection 10ml + 5% glucose injection 50ml intravenous drip bid, used during the interval of DMPS withdrawal; ③ Zinc gluconate tablets 280mg orally tid.

[0243] Observation group: conventional western medicine + Bushen Huoxue Huazhuo prescription (6 courses of treatment): on the basis of the control group, Chinese medicine Bushen Huoxue Huazhuo prescription (20g of Rehmannia glutinosa, 15g of Morinda officinalis, 6g of Schisandra chinensis, 15g of Salvia miltiorrhiza, 9g of Coptis chinensis, 9g of Alisma orientalis, 9g of Curcuma zedoaria, 6g of Rhubarb, 15g of Lysimachia chinensis, 10g of Curcuma longa) was added. Each dose was decocted in 150ml of water and taken in the morning and evening.

[0244] Key drug information

[0245]

[0246] 2.2 Observation indicators

[0247] (1) Changes in clinical efficacy

[0248] The clinical efficacy of the two groups of patients after treatment was observed.

[45] The symptoms and signs were divided into markedly effective, improved, ineffective, and worsened (see Appendix 1).

[0249] (2) Changes in UWDRS scores

[0250] The changes of Unified Wilson Disease Rating Scale (UWDRS) scores were observed in the two groups of patients before and after treatment. The first part of the scale is the neurological function score, including 27 parts, totaling 108 points; the second part is the liver function score, including 9 parts, totaling 36 points; the third part is the mental symptom score, including 19 parts, totaling 76 points. The higher the score, the more severe the symptoms (see Appendix 2).

[0251] (3) Changes in TCM syndrome score

[0252] The changes of TCM syndrome scores in the two groups were observed before and after treatment.

[0253] (4) Laboratory examination

[0254] The 24-hour urine copper, serum NfL and SOD levels of patients were tested before and after treatment. The specific operation methods for the determination of serum NfL and SOD levels are detailed in "Part I". The 24-hour urine copper was tested by the Laboratory Department of Anhui University of Traditional Chinese Medicine.

[0255] (5) Safety indicators

[0256] During the entire medication period, the patient's general examination items were observed, including basic vital signs, blood, urine, stool routine tests, liver and kidney function, etc., and adverse events were recorded and reported in a timely manner.

[0257] 2.3 Statistical analysis

[0258] SPSS 26.0 statistical software will be used for statistical analysis, and data will be expressed as x±s. Independent sample t test will be used when two groups of independent and continuous numerical variable data meet normal distribution. Statistical results P < 0.05 indicates statistically significant differences (* above the numbers in the chart represents P < 0.05, ** represents P < 0.01, and *** represents P < 0.001).

[0259] 3 Results

[0260] 3.1 Comparison of general clinical data of research subjects

[0261] A total of 58 patients with WD liver and kidney deficiency and phlegm and blood stasis were included in this study, with 29 cases in the observation group and 29 cases in the control group. There were 16 males and 11 females in the control group, with an average age of 27.38±6.81 years; there were 14 males and 15 females in the observation group, with an average age of 28.24±8.20 years. As shown in Table 6, Figure 8 There was no significant difference in the comparison of clinical general data between the control group and the Bushen Huoxue Huazhuo prescription combined with western medicine group (P>0.05).

[0262] Table 6 Comparison of general clinical data

[0263]

[0264] 3.2 Comparison of clinical efficacy between the two groups of patients after treatment

[0265] As shown in Table 7, Fig. 9 As shown in the figure, after the sixth course of treatment, the overall effective rate of the observation group was 79.31%, including 4 markedly effective patients, 19 improved patients, 5 ineffective patients, and 1 deteriorated patient; the overall effective rate of the control group was 51.72%, including 2 markedly effective patients, 13 improved patients, 10 ineffective patients, and 4 deteriorated patients. The chi-square test was used to compare the data of the two groups, P = 0.027 (P < 0.05), which was statistically significant, indicating that the clinical effect of the Bushen Huoxue Huazhuo prescription combined with western medicine in the treatment of WD is better than that of western medicine alone to remove copper.

[0266] Table 7 Comparison of overall effective rate between the observation group and the control group

[0267]

[0268] Note: * Compared with the control group, P<0.05.

[0269] 3.3 Comparison of UWDRS scores between the two groups

[0270] As shown in Table 8, Fig.10As shown in the data, there was no significant difference in UWDRS scores between the two groups of patients before treatment (P=0.584>0.05). The UWDRS scores of the two groups of patients decreased significantly after treatment (P<0.05). After treatment, the comparison between the two groups found that the UWDRS score of the Bushen Huoxue Huazhuo prescription combined with western medicine group decreased more significantly (P<0.01).

[0271] Table 8 Comparison of UWDRS scores between the two groups before and after treatment

[0272]

[0273] 3.4 Comparison of TCM syndrome scores between the two groups

[0274] From Table 9, Fig.11 It can be seen that there was no significant difference in the TCM syndrome scores between the two groups of patients before the start of the experiment (P=0.2983>0.05), and the TCM syndrome scores of the two groups decreased significantly after treatment (P<0.01); after treatment, the inter-group comparison showed that the TCM syndrome score of the Bushen Huoxue Huazhuo prescription combined with western medicine group decreased more significantly (P<0.01).

[0275] Table 9 Comparison of TCM syndrome scores between the two groups of patients before and after treatment

[0276]

[0277] 3.5 Comparison of 24-hour urinary copper between the two groups

[0278] There was no significant difference in 24-hour urinary copper before treatment between the control group and the Bushen Huoxue Huazhuo prescription combined with western medicine group (P>0.05); the 24-hour urinary copper in the Bushen Huoxue Huazhuo prescription combined with western medicine group increased significantly after treatment (P<0.01), and the 24-hour urinary copper in the control group increased slightly after treatment (P<0.05); after treatment, the comparison between the two groups showed that the 24-hour urinary copper in the Bushen Huoxue Huazhuo prescription combined with western medicine group increased more significantly, and the copper removal effect was better (P<0.05). See Table 10 for details. Fig.12 .

[0279] Table 10 Comparison of 24h urine copper (μg) between the two groups of patients before and after treatment

[0280]

[0281] 3.6 Comparison of serum NfL between the two groups

[0282] There was no significant difference in serum NfL values ​​between the two groups before treatment (P>0.05); after treatment, the serum NfL values ​​of the observation group and the control group decreased significantly compared with those before treatment (P<0.01); after treatment, the serum NfL values ​​of the Bushen Huoxue Huazhuo prescription combined with western medicine group decreased more significantly than those of the control group (P<0.01). See Table 11 for details. Fig.13 .

[0283] Table 11 Comparison of serum NfL (pg / mL) levels between the two groups of patients before and after treatment

[0284]

[0285] 3.7 Comparison of serum SOD between the two groups

[0286] There was no significant difference in serum SOD between the two groups of patients before treatment (P>0.05); serum SOD of the observation group increased significantly after treatment (P<0.01), and serum SOD of the control group increased slightly after treatment (P<0.05); after treatment, the two groups were compared, and the Bushen Huoxue Huazhuo prescription combined with western medicine group was significantly higher than the control group (P<0.01). See Table 12 for details. Fig.14 .

[0287] Table 12 Comparison of serum SOD (U / mL) levels between the two groups of patients before and after treatment

[0288]

[0289] 4 Discussion

[0290] 4.1 Understanding of WD in Traditional Chinese Medicine

[0291] According to the clinical manifestations of WD, such as abdominal distension, slow movement, yellowing of the body and eyes, tremor of limbs, etc., Chinese medicine can classify it as "liver wind", "tremor disease", "accumulation" and other diseases. In terms of the etiology and pathogenesis of Chinese medicine, WD is a hereditary disease, and the damage of congenital essence is the root cause of the disease. According to the etiology and pathogenesis of WD, its treatment method is mainly based on clearing away heat and dampness, promoting qi and removing phlegm, and promoting blood circulation and removing blood stasis, but at the same time, attention should be paid to the importance of nourishing the liver and kidney in its treatment.

[0292] 4.2 Western medicine’s understanding of WD

[0293] WD is inherited in an autosomal recessive manner. Dysfunction of the ATP 7B protein causes cell damage, and large amounts of copper are excreted into the bile, which in turn leads to copper accumulation in multiple organ systems. Although the phenotype varies greatly, the typical manifestation is liver disease and movement disorders in adolescence or early adulthood. Most patients are divided into neurological and liver types based on the type of manifestation. The diagnosis of WD usually requires a combination of biochemical test results (low serum cerebrospinal fluid concentration, low serum total copper concentration, and increased urinary copper excretion) and / or the discovery of a double pathogenic variant (or possible pathogenic variant) in the ATP 7B gene through molecular genetic testing. If WD is not treated in time, it may lead to severe disability and death. Its diagnosis is easily overlooked, but if it is discovered early, effective treatment can prevent or reverse many manifestations of this disease. Treatment of WD includes lifelong oral medication and avoiding copper intake in the diet. If treatment is started early and medication is taken consistently, the survival rate of WD patients is similar to that of the general population. Its main therapeutic drugs include copper chelator D-penicillamine, sodium dimercaptopropane sulfonate (DMPS), trientine, and zinc that inhibits intestinal copper absorption. Penicillamine is a cysteine-rich protein that can enhance urinary copper excretion and maintain copper homeostasis in the body by inducing metallothionein. DMPS is a chelator containing two thiol groups that was first used in WD in my country. It has good water solubility and is currently the most commonly used intravenous copper chelator. Its side effects are smaller than those of penicillamine and its therapeutic effect is better. Zinc is a treatment alternative to penicillamine and tamoxifen. It can be used as a maintenance therapy or as a first-line treatment, usually for asymptomatic patients. Zinc mainly inhibits the absorption of dietary copper by inducing the expression of endogenous copper chelator metallothionein in the intestine and liver, and is relatively well tolerated.

[0294] 4.3 The basis and analysis of the formula for the Bushen Huoxue Huazhuo prescription

[0295] The prescription for nourishing the liver and kidney, promoting blood circulation and removing turbidity is composed of the following medicines: Rehmannia glutinosa, Morinda officinalis, Schisandra chinensis, Salvia miltiorrhiza, Coptis chinensis, Rhizoma Zedoariae, Curcuma zedoariae, Rhubarb, Herba Lysimachiae, and Curcuma longa. The main medicines are Rehmannia glutinosa, Morinda officinalis, and Schisandra chinensis. The three medicines are used together to nourish the liver and kidney, benefit the essence, and replenish the liver and blood; Herba Lysimachiae, Rhizoma Zedoariae, and Coptis chinensis clear away heat and dampness, clear the bowels and remove turbidity, and Curcuma zedoariae and Curcuma longa can promote qi and promote blood circulation, and are the ministerial medicines; Rhubarb can clear away heat and toxicity, break up stagnation, and promote blood stasis as the adjuvant. The whole prescription can not only nourish the liver and kidney, clear the bowels and remove turbidity, and accelerate the excretion of copper, but also remove dampness and heat in the body, promote qi and promote blood circulation, and improve the accumulation of blood stasis and qi stagnation in the body.

[0296] 4.4 Analysis of the clinical efficacy of the prescription for tonifying the kidney, activating blood circulation and removing turbidity

[0297] This study found that the overall effective rate of Bushen Huoxue Huazhuo prescription combined with western medicine for copper removal was 79.31%, while the overall effective rate of the control group using western medicine alone for copper removal was 51.32%. The total effective rate after treatment was significantly higher in the Bushen Huoxue Huazhuo prescription combined with western medicine group than in the control group (P < 0.05). In terms of the TCM syndrome score, the Bushen Huoxue Huazhuo prescription combined with western medicine group and the control group were significantly lower after treatment than before treatment (P < 0.01). After treatment, the TCM syndrome score of the Bushen Huoxue Huazhuo prescription combined with western medicine group decreased more significantly (P = 0.0036 < 0.01). Compared with the UWDRS score before treatment, the average score of the observation group after treatment was significantly lower than that of the control group (16.97 ± 13.89 VS 25.41 ± 16.39, P = 0.0387 < 0.01). In terms of 24-hour urinary copper, the 24-hour urinary copper of the observation group increased significantly after treatment (P < 0.01), and the 24-hour urinary copper of the control group increased slightly after treatment (P < 0.05); after treatment, the increase in 24-hour urinary copper in the Bushen Huoxue Huazhuo prescription combined with western medicine copper removal group was more obvious, and the copper removal effect was better (P < 0.05). The above results all indicate that the overall clinical effect of Bushen Huoxue Huazhuo prescription combined with conventional copper removal drugs in the treatment of WD patients is better than that of conventional copper removal western medicine alone, and its mechanism of action is speculated to be related to the cubic basis and ideas of this study.

[0298] 4.5 Analysis of the effects of the Bushen Huoxue Huazhuo prescription on serum NfL and SOD

[0299] Studies have shown that neurofilaments, in addition to being structural elements in axons, can also form cell scaffolds for docking and organizing synaptic vesicles, endosomes, and endoplasmic reticulum (ER). The radial growth and structural stability of myelinated axons and the optimal propagation speed (conduction velocity) of electrical impulses along axons are inseparable from NF. NfL, as the lightest, highest content, and most soluble subtype of NF, is only expressed in neurons, so it has high sensitivity and specificity for detecting neuronal damage and death. This study found that after treatment, the serum NfL levels of the Bushen Huoxue Huazhuo prescription combined with Western medicine group and the control group were significantly decreased compared with those before treatment (P < 0.01), and the serum NfL levels of the Bushen Huoxue Huazhuo prescription combined with Western medicine group decreased more significantly than those of the control group (P < 0.01). This shows that the Bushen Huoxue Huazhuo prescription can effectively improve the clinical symptoms of patients with WD liver and kidney deficiency and phlegm and blood stasis syndrome by repairing nerve axonal damage and stabilizing neuronal structure.

[0300] When the level of intracellular free Cu is too high, it will promote the production of reactive oxygen through the Fenton reaction and increase the oxidative stress response. Superoxide dismutase (SOD) is a metal enzyme in the human body that can detoxify superoxide free radicals and is expressed in a variety of tissues (including the brain). It has the function of regulating the balance of oxidation and anti-oxidation in the body and protecting the body from oxidative stress damage. This study found that compared with the same group before treatment, the serum SOD of the observation group was significantly increased after treatment (P < 0.01), and the serum SOD of the control group was slightly increased after treatment (P < 0.05); after treatment, the two groups were compared, and the Bushen Huoxue Huazhuo prescription combined with Western medicine group was significantly higher than the control group (P = 0.0093 < 0.01). This shows that the Bushen Huoxue Huazhuo prescription can improve the microenvironment in the body, reduce the toxic side effects of oxidative stress, promote the excretion of copper toxicity, and improve the clinical efficacy.

[0301] 4.6 Test conclusion

[0302] 1. Serum NfL can be used as a new biomarker for the diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type, while serum SOD can only be used as a potential biomarker for the diagnosis of hepatolenticular degeneration. Both can reflect the degree of progression of hepatolenticular degeneration and serve as indicators for judging clinical efficacy.

[0303] 2. The Bushen Huoxue Huazhuo prescription combined with Western medicine for copper removal can effectively improve the clinical symptoms of hepatolenticular degeneration with liver and kidney deficiency and phlegm and blood stasis, and can significantly reduce serum NfL activity and increase SOD expression level. It is speculated that it is related to potential mechanisms such as repairing nerve axonal damage, scavenging free radicals, and reducing oxidative stress response in the body. On the other hand, this experiment can indirectly illustrate that from the perspective of therapeutic medication, serum NfL can be used as a new biomarker for the diagnosis of hepatolenticular degeneration and the differential diagnosis of hepatic and cerebral types, and serum SOD can only be used as a potential biomarker for the diagnosis of hepatolenticular degeneration.

Claims

1. A biomarker for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type, characterized by The biomarkers include serum neurofilament light chain protein NfL.

2. The biomarker for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to claim 1, characterized in that The biomarkers also include superoxide dismutase SOD.

3. The method for testing and verifying biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to any one of claims 1 to 2, characterized in that include: Test 1: Serum neurofilament light chain protein and superoxide dismutase for early diagnosis of Wilson disease and differential diagnosis of hepatic and cerebrovascular type: (1) Collect patient information (2) Serum neurofilament light chain protein NfL detection 2-1) Sample collection and preservation All subjects were given 3-5 ml of fasting venous blood from patients and healthy subjects in the morning using serum tubes. The collected blood samples were placed at room temperature for 2 hours, then centrifuged at 1000×g for 20 minutes at 4°C, the upper suspended matter was removed, and the supernatant was placed at -80°C for testing. The serum samples were tested using the enzyme-linked immunosorbent assay (ELISA) method. 2-2) Operation steps The fasting venous blood of the patients and healthy subjects was centrifuged in the morning for testing. The specific testing steps are as follows: ① Take out the required strips from the aluminum foil bag after equilibration at room temperature for 10 minutes, and seal the remaining strips in a ziplock bag and place them back at 4℃; ② Sample addition: add 100 μL of samples or standards of different concentrations to the corresponding wells, add 100 μL of universal diluent to the blank wells, cover with sealing film and incubate at 37°C for 60 minutes; ③ Add biotinylated antibody: Take out the enzyme label solution, discard the liquid, do not wash, directly add 100 μL of biotinylated antibody working solution to each well, cover with sealing film and incubate at 37°C for 60 minutes; ④ Wash the plate: discard the liquid, add 300μL 1× washing solution to each well, let it stand for 1 minute, shake off the washing solution, pat dry on absorbent paper, and repeat washing the plate 3 times; ⑤ Add enzyme conjugate working solution: Add 100 μL of enzyme conjugate working solution to each well, cover with sealing film and incubate at 37°C for 30 minutes; ⑥ Wash the plate: discard the liquid and wash the plate 5 times according to the washing method in step ④; ⑦ Add substrate: Add 90 μL of substrate TMB3,3',5,5'-tetramethylbenzidine to each well, cover with sealing film, and incubate at 37°C in the dark for 15 minutes; ⑧Add stop solution: Take out the ELISA solution or ELISA plate, add 50 μL of stop solution directly to each well, and immediately measure the OD value of each well at a wavelength of 450 nm; (3) Serum superoxide dismutase (SOD) detection; 3-1) Sample collection and storage are the same as "2-1) Sample collection and storage"; 3-4) Operation steps The fasting venous blood of the patients and healthy subjects was centrifuged in the morning for testing. The specific testing steps are as follows: ① Dilution and addition of standard: set 10 wells for standard, add 50μL of standard diluent, mix and dilute; ② Sample addition: Set up blank wells and sample wells respectively. First add 40 μL of sample diluent to the sample wells on the enzyme-labeled plate, and then add 10 μL of the sample to be tested. The final dilution of the sample is 5 times; ③ Add enzyme: add 50 μL of enzyme labeling reagent to each well, except for the blank well; ④ Incubation: Seal the plate with a sealing film and incubate at 37°C for 30 minutes; ⑤ Liquid preparation: dilute the 30 times concentrated washing solution with 30 times distilled water for later use; ⑥ Washing: Carefully peel off the sealing film, discard the liquid, spin dry, fill each well with washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry; ⑦ Color development: first add 50μL of color developer A to each well, then add 50μL of color developer B, gently shake and mix, and color development at 37℃ in the dark for 10 minutes; ⑧Stop: Add 50 μL of stop solution to each well to stop the reaction, and the blue color will turn yellow immediately; ⑨ Use the blank well as zero setting, and measure the absorbance OD value of each well in sequence at a wavelength of 450nm; (4) Statistical analysis ① The experimental data were statistically analyzed using SPSS 26.0 statistical software: the independent sample t test was used to compare the two independent numerical variable data when they met the normal distribution; the chi-square test was used to analyze the correlation and difference of the completely random unordered categorical variable data. 2 test; The two groups refer to the WD-brain type group and the WD-liver type group in the general data, which are two independent numerical variable data in the comparison of disease course; the WD-brain type group and the WD-liver type group in the general data are completely random and unordered categorical variable data in the comparison of gender; ②When the three groups of samples meet normality, homogeneity of variance and independence at the same time, one-way analysis of variance is used; If not satisfied, the Kruskal-Wallis H rank sum test was used; ③ The receiver operating characteristic curve (ROC) analysis was used to evaluate the predictive efficacy of serum NfL and SOD expression levels in the diagnosis of WD patients and the differential diagnosis of hepatocerebral type, calculate the sensitivity and specificity, and evaluate the authenticity of the prediction; (5) Test results 5-1) Comparison of general clinical data A total of 60 patients with Wilson's disease who met the inclusion criteria were selected, including 24 patients with hepatic type and 36 patients with brain type. During the process, there was no dropout in the hepatic type group, 2 patients in the brain type group, 1 patient withdrew from the experiment due to an accidental fall, and 1 patient refused to do a routine stool test; there were 30 healthy control groups with no dropouts; the actual enrolled patients were 24 patients in the WD-hepatic type group and 34 patients in the WD-brain type group; there were 16 males and 14 females in the healthy control group, with an average age of 27.43±5.66 years old; there were 19 males and 15 females in the WD-brain type group, with an average age of 28.72± 7.92 years old, with an average course of disease of 11.94±5.345 years; 13 males and 11 females in the WD-liver type group, with an average age of 27.67±8.53 years old and an average course of disease of 10.83±3.841 years; The chi-square test was used to analyze the gender of the three groups of patients and analyze their statistical significance. The age and course of disease were tested for normality and homogeneity of variance to determine whether the observation period met the normal distribution and homogeneity of variance. The age of the three groups was analyzed by one-way analysis of variance. The course of disease in the WD-liver type group and the WD-brain type group was tested by independent sample t test, and the results were listed and plotted at the same time; 5-2) Differences in the expression of serum neurofilament light chain protein NfL among the three groups The serum NfL levels among the three subgroups were statistically analyzed. First, a normality test was performed, and it was found that none of the three groups conformed to the normal distribution. Therefore, the Kruskal-Wallis H test was used to compare the three groups, including the comparison of relevant indicators among the three groups: WD-liver type, WD-brain type and healthy control group; then the Nemenyi test was used to perform post hoc comparisons between the two groups, and the tables and graphs were observed at the same time; 5-3) Differences in the expression of serum superoxide dismutase (SOD) levels among the three groups Similarly, the Kruskal-Wallis H test was used to detect the serum superoxide dismutase SOD levels of the three groups, and the results were tabulated and plotted; 5-4) ROC curve analysis In order to evaluate the value of the two indicators for the diagnosis of WD and the differential diagnosis of hepatocerebral type, ROC curve analysis was performed to observe the cutoff value, sensitivity, specificity, and area under the AUC curve of WD patients compared with the healthy control group, and the specific ROC curve of serum NfL level expression was plotted; including the cutoff value, sensitivity, specificity, and area under the AUC curve between WD hepatocerebral types, and the plot was also plotted; If the statistical results have proved that there is no significant difference in serum SOD levels between WD brain-type patients and WD liver-type patients, the ROC curve diagram of serum SOD level expression can be drawn only for WD patients and healthy controls; 5-5) Correlation analysis between serum NfL, SOD and age, disease duration and UWDRS score If serum NfL does not obey the normal distribution, the Spearman correlation coefficient can be used to analyze the correlation between serum NfL and age, disease duration and UWDRS score. At the same time, the correlation between serum NfL and age, disease duration and UWDRS score can be listed and analyzed graphically.

4. The method for testing and verifying biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to claim 3, characterized in that: The above (1) collecting patient information includes: Name, gender, disease course, clinical symptoms, family history, laboratory test indicators, UWDRS scale test; observation indicators include: serum neurofilament light chain protein NfL, superoxide dismutase SOD.

5. The test method for biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to claim 1, characterized in that: 2-2) Operation steps: The diluent is a universal diluent, 96-well configuration: 2×20mL; 48-well configuration: 1×20mL; storage conditions after opening: 2-8°C; The concentrated biotinylated antibody 100× Biotin-antibody (100×) is 120 μL for 96-well configuration and 60 μL for 48-well configuration. Preparation of the biotinylated antibody working solution: 15 minutes before use, centrifuge the concentrated biotinylated antibody at 1000×g for 1 minute, dilute the 100× concentrated biotinylated antibody to 1× working concentration with universal diluent, and prepare it for use immediately; The concentrated enzyme binding working solution 100×Streptavidin-HRP (100×), 96-well configuration: 120 μL; 48-well configuration: 60 μL; storage conditions after opening: -20°C, away from light; Preparation of the enzyme conjugate working solution: 15 minutes before use, centrifuge the 100× concentrated enzyme conjugate at 1000×g for 1 minute, dilute the 100× concentrated HRP enzyme conjugate to 1× working concentration with universal diluent, and prepare it for use immediately.

6. The test method for biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to claim 1 or 5, characterized in that: The universal diluent: 96-well configuration: 2×20 mL; 48-well configuration: 1×20 mL; storage conditions after opening: 2-8°C.

7. The test method for biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to claim 1, characterized in that: 3-4) Operation steps: the color developing agent solution is used as a substrate of horseradish peroxidase HRP in ELISA, and the color developing agent is TMB3,3',5,5'-tetramethylbenzidine.

8. The test method for biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to claim 1, characterized in that Also includes: Experiment 2: Clinical observation on the effect of Bushen Huoxue Huazhuo prescription on the expression levels of serum NfL and SOD in patients with liver-kidney deficiency and phlegm-stasis syndrome 1 Test subjects 1.1 Case source A total of 58 WD patients who met the inclusion criteria were selected and randomly divided into an observation group and a control group at a ratio of 1:1, with 29 cases in each group; the control group had 16 males and 11 females; the observation group had 14 males and 15 females; 2 Test content 2.1 Group treatment Control group: 6 courses of conventional western medicine, 6 days per course, 2 days off: conventional copper excretion drugs: ① 5% glucose injection 250ml + dithiothiopropane sulfonate injection DMPS 0.02g / kg·d intravenous drip qd; ② 10% calcium gluconate injection 10ml + 5% glucose injection 50ml intravenous drip bid, used during the interval of DMPS withdrawal; ③ zinc gluconate tablets 280mg orally tid; Observation group: conventional western medicine + Bushen Huoxue Huazhuo prescription, Bushen Huoxue Huazhuo prescription was used for 6 courses, each dose of crude drug weighed 200g, taken in the morning and evening: on the basis of the control group, Chinese medicine Bushen Huoxue Huazhuo prescription was added: Rehmannia glutinosa 20g, Morinda officinalis 15g, Schisandra chinensis 6g, Salvia miltiorrhiza 15g, Coptis chinensis 9g, Alisma orientalis 9g, Curcuma 9g, Rhubarb 6g, Lysimachia chinensis 15g, Curcuma longa 10g; 2.2 Observation indicators (1) Changes in clinical efficacy The clinical efficacy of the two groups of patients after treatment was observed. According to the modified Goldstein clinical grading standard, the symptoms and signs were divided into markedly effective, improved, ineffective, and worsened. (2) Changes in UWDRS scores The Wilson disease rating scale was observed in both groups before and after treatment. (3) Changes in TCM syndrome score Observe the changes of TCM syndrome scores in the two groups before and after treatment; (4) Laboratory examination The patients' 24-hour urine copper, serum neurofilament light chain protein NfL, and superoxide dismutase SOD levels were tested before and after treatment. The specific operation methods for the determination of serum NfL and SOD levels are detailed in Experiment 1 (2) Serum neurofilament light chain protein NfL detection (3) Serum superoxide dismutase SOD detection; (5) Safety indicators Observe the patient's general examination items during the entire medication period, including basic vital signs, blood, urine, stool routine tests, and liver and kidney function, and record and report adverse events in a timely manner; 2.3 Statistical analysis SPSS 26.0 statistical software will be used for statistical analysis. Indicates; when two groups of independent and continuous numerical variable data meet the normal distribution, the independent sample t test is used; the statistical result P<0.05 indicates that the difference is statistically significant; 3 Test results 3.1 Comparison of general clinical data A total of 58 patients with WD liver and kidney deficiency and phlegm and blood stasis were included, with 29 cases in the observation group and 29 cases in the control group. There were 16 males and 11 females in the control group, with an average age of 27.38±6.81 years; there were 14 males and 15 females in the observation group, with an average age of 28.24±8.20 years; 3.2 Comparison of clinical efficacy between the two groups of patients after treatment The overall effective rate of the observation group and the control group after the sixth course of treatment were compared; the P value of the two groups was compared by chi-square test; 3.3 Comparison of UWDRS scores between the two groups The UWDRS scores of the two groups of patients before and after treatment were compared; 3.4 Comparison of TCM syndrome scores between the two groups of patients The TCM syndrome score values ​​of the two groups of patients were observed before the experiment and after treatment. The TCM syndrome score values ​​of the two groups were compared after treatment, and the TCM syndrome score values ​​of the Bushen Huoxue Huazhuo prescription combined with western medicine group were observed. 3.5 Comparison of 24-hour urinary copper between the two groups Compare the urinary copper in the control group and the Bushen Huoxue Huazhuo prescription combined with western medicine group before treatment, and the difference of urinary copper between the two groups after treatment; 3.6 Comparison of serum neurofilament light chain protein NfL between the two groups of patients Comparison of serum NfL values ​​between the two groups before treatment; Comparison of serum NfL values ​​between the observation group and the control group after treatment; Comparison of serum NfL values ​​between the two groups after treatment, between the Bushen Huoxue Huazhuo prescription combined with western medicine group and the control group; 3.7 Comparison of serum superoxide dismutase (SOD) between the two groups The difference in serum SOD between the two groups of patients before treatment was observed after statistical processing; the serum SOD level of the observation group after treatment, the serum superoxide dismutase SOD level of the control group after treatment; the two groups were compared after treatment, and the difference between the Bushen Huoxue Huazhuo prescription combined with western medicine group and the control group was compared.

9. The test method for biomarkers for diagnosis of hepatolenticular degeneration and differential diagnosis of hepatocerebral type according to claim 8, characterized in that: When testing serum NfL levels, place the whole blood sample collected in the serum separation tube at room temperature for 2 hours or at 2-8°C overnight, then centrifuge at 1000×g for 20 minutes and take the supernatant, or store the supernatant at -20°C or -80°C, but avoid repeated freezing and thawing; Preparation work before testing serum NfL levels: add 1mL of universal diluent to the freeze-dried standard, let it stand for 15 minutes until it is completely dissolved, then gently mix it, the concentration is 500pg / mL, and finally determine the following concentrations: 500pg / mL, 250pg / mL, 125pg / mL, 62.5pg / mL, 31.2pg / mL, 15.6pg / mL, 7.8pg / mL, 0pg / mL for dilution, take 7 EP centrifuge tubes, add 500μL of universal diluent to each tube, draw 500μL from the 500pg / mL standard working solution into the first EP tube and mix it to make a 250pg / mL standard working solution, and follow this step to draw and mix in sequence; the last tube is directly used as a blank well, and there is no need to draw liquid from the second to last tube; During the sample addition process for serum NfL level detection, samples or standards of different concentrations were added to the corresponding wells at 100 μl per well, and 100 μl of universal diluent was added to the blank wells.

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